Materials transport device for diagnostic and tissue engineering applications

ABSTRACT

Devices that can transport biological materials are described. The devices incorporate capillary channeled fibers that can effectively transport living cells as well as other biological materials such as nutrients, growth factors, waste materials, etc. The devices can include a sorptive material at one end of the fibers that can improve transport of materials through the devices. The devices can differentially transport different cell types, particularly when the fibers are held in a vertical orientation. Diagnostic devices that incorporate the capillary channeled fibers are described that can be utilized to separate cell types from one another. Tissue engineering scaffolds that incorporate the capillary channeled fibers are described that can more efficiently transport materials into and out of the scaffolds.

CROSS REFERENCE TO RELATED APPLICATION

This application claims filing benefit of U.S. Provisional PatentApplication Ser. No. 61/904,167 having a filing date of Nov. 14, 2013,which is incorporated herein in its entirety by reference.

FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This invention was made with government support under contract numberW81XWH-05-1-0379 and NSF Grant No. 0736007, awarded by the U.S.Department of Defense. The government has certain rights in theinvention.

BACKGROUND

Efficient methods to identify and evaluate particular cell types wouldbe useful in a wide variety of applications. For instance, cellulardiagnostic testing, e.g., the diagnosis of disease states based upon thepresence or absence of certain cell types, ideally utilizes testingprotocols that can provide both speed and accuracy, as well as ease ofuse. By way of example, cancer, which is the world's deadliest and mostcostly disease requires the accurate recognition of the presence ofcancer cells for proper diagnosis. Moreover, as early diagnosis can bekey in improved survival rates, testing protocols that can recognizecancer cells at very low concentration are of great benefit.

While current cancer testing protocols such as histology/cytologytesting, immunoassays, flow cytometry, and so forth have improved cancerscreening and cancer survival rates, room for improvement in the artexists. For example, many testing protocols require multiple expensivetests to make a confident diagnosis, and protocols often lack highreliability. Testing methods are often complicated and require complexprocedures that can increase prices. In addition, testing often isinadequate to distinguish the state of a cancer, for instance failing indistinguishing benign cancer cells from malignant cancer cells. Thus, areliable and inexpensive device and method that could identify diseasedcells in a test sample would be of great benefit.

The ability to quickly and reliably identify particular cell typesand/or subpopulations of particular cell types would be of benefit inother applications as well. For instance, a major limitation indeveloping tissue engineered constructs is the ability to identify andisolate functional cells, typically progenitor cells, from the patientor donor. The ability to reliably and accurately test the function andthe cellular make-up of transplant tissue would also be of great benefitto, e.g., prevent the transplant of diseased tissue. Conventionalmethods to identify and isolate desirable functional cells involve usingcell surface markers and morphological traits as well as cell platingand cell adherence methods. However, many cell types lack common markersor universal morphological traits, making reliable testing extremelydifficult. Conventional methods are expensive and do not directlyevaluate cell function.

The development of engineered tissue constructs faces otherdifficulties, in addition to identification and utilization of desiredprogenitor cells. For example, there are currently constraints on thesize of tissue constructs that can be created, and a major shortcomingof tissue engineering is that the size of the grafts that can begenerated is small relative to the size of the defects that they aremeant to treat. For example, autologous osteochondral grafting ormosaicplasty, often referred to as the gold standard for the treatmentof cartilage defects and osteoarthritis, fails in the therapy of largelesions measuring more than about 20 millimeters in diameter, and theviable constructs formed to date are considerably smaller than thissize.

The size constraints that tissue engineering currently faces are largelydue to the non-homogeneous growth of cells on the traditional ‘porousblock’ scaffolds, which prevents the formation of a functional constructfrom surface to core. This is due to two major limiting factors: lack ofuniform cell seeding though the entire thickness of the scaffold and themass transfer limitations of nutrients and waste removal. Because of thecomplex architecture of many 3D scaffolds, dispersing a high density ofcells with high efficiency and uniformity throughout the scaffold volumeis difficult. An additional factor in the survival of thick 3Dengineered tissues is the delivery of oxygen and nutrients to the entireconstruct, and waste removal from the construct. The movement ofnutrients to the cells and removal of waste products from the cells hasto rely on molecular diffusion due to the lack of a vascular system. Assuch, nutrients are depleted before reaching the inner core of theconstruct and waste products accumulate. Cells that migrate into thecore become necrotic and the living cell population is commonlyconcentrated at the periphery of the scaffold. There is also a delay intissue formation or lack of tissue formation in the center of thescaffold due to the lack of biomolecule/growth factor penetration. Anexample commonly occurs in posterolateral fusions there is a “lageffect” of bone formation in the center of the scaffold.

What are needed in the art are devices and methods that can transportbiological materials effectively. For instance, a device that cantransport different cell types at different rates would be beneficial toprovide a low cost, fast, and accurate cell recognition protocol.Additionally, a device that can transport biological materials such ascells, nutrients, biomolecules, and waste in a fast and efficient mannerwould be of benefit in tissue engineering.

SUMMARY

According to one embodiment, disclosed is a device for transportingbiological materials. The device includes a plurality of capillarychanneled fibers that are adjacent to one another and generally alignedwith one another in a bundle. Each of the capillary channeled fibersincludes a plurality of co-linear channels extending along the length ofthe fiber, each channel being defined by a pair of opposed walls thatextend longitudinally along the length of the fiber with the opposedwalls forming a part of the exterior surface of the fiber. The fibersare held adjacent to one another such that at least one channel isformed between two adjacent capillary channeled fibers of the bundle.

The device also includes a cap that covers one end of the bundle. Morespecifically, a first end of the bundle of capillary channeled fibers isenclosed within the cap and the cap can include a sorptive material thatsurrounds the first end of the bundle.

According to another embodiment, disclosed is a diagnostic device. Thediagnostic device includes a well that is capable of holding a fluidsample. The diagnostic device also includes a capillary channeled fiberand a brace that secures the capillary channeled fiber in a verticalorientation with a first end of the fiber within the well. For instance,in one embodiment, the brace can be a lid that sits over the well. Thelid can include an aperture that passes from a first side to a secondside of the lid, and the fiber can be held securely by the lid in avertical orientation with one end of the fiber within the well.

In one embodiment, the fiber of the diagnostic device can be a member ofa bundle of fibers that include capillary channeled fibers. The fiberscan be held adjacent to one another and generally aligned with oneanother such that at least one channel is formed between two adjacentcapillary channeled fibers of the bundle. In yet another embodiment, thefiber bundle of the diagnostic device can include a cap that covers oneend of the bundle. More specifically, a first end of the bundle can beheld within the well and a second end of the bundle can be enclosedwithin the cap and the cap can include a sorptive material thatsurrounds the first end of the bundle.

According to another embodiment, disclosed is a tissue engineeringscaffold. The tissue engineering scaffold includes a porous matrix of abiocompatible material that can support cell growth and development andalso includes a device for transporting biological materials. The devicefor transporting biological materials includes the bundle of capillarychanneled fibers and a cap on one end of the bundle; the cap including asorptive material that surrounds the end of the bundle of fibers. Atleast a portion of the bundle of capillary channeled fibers can beenclosed within the porous matrix. The cap that covers one end of thebundle of fibers can be within the porous matrix in one embodiment, andcan be external to the porous matrix in another embodiment.

Also disclosed are methods for separating biological materials such ascells or DNA from other materials by use of the devices. For instance, amethod can include locating a first end of a materials transport deviceas disclosed herein within a solution that includes a first cell and asecond cell, holding the first end of the fiber (which correlates withthe first end of the device) within the solution for a period of time,the first cell wicking through the channels of the fiber to a firstlocation of the materials transport device during the period of time andthe second cell wicking through the channels of the fiber to a secondlocation of the materials transport device during the period of time.The method can also include recovering at least one of the first cell orthe second cell from the materials transport device.

BRIEF DESCRIPTION OF THE FIGURES

The presently disclosed subject matter may be better understood withreference to the Figures, of which:

FIG. 1 illustrates a bundle of two capillary channeled fibers as may beincorporated in a materials transport device as disclosed herein.

FIG. 2 illustrates the cell separation capabilities of a materialstransport device as disclosed herein. FIG. 2A illustrates a completefiber bundle following separation of cells and FIG. 2B illustrates thefiber bundle separated into a top region and a bottom region.

FIG. 3 is a schematic illustration of a diagnostic device employing thematerials transport device.

FIG. 4 illustrates a cross sectional view of bundled capillary channeledfibers.

FIG. 5 illustrates one embodiment of a materials transport device asdisclosed herein.

FIG. 6 illustrates the cell separation capability of a materialstransport device as illustrated in FIG. 5.

FIG. 7 is a schematic illustration of a production process as may beused to produce a tissue engineering scaffold as described herein.

FIG. 8 is a schematic illustration of a tissue engineering scaffoldincorporating a materials transport device as disclosed herein.

FIG. 9 illustrates the displacement of cancer and normal cells on adiagnostic device as described herein.

FIG. 10 graphically illustrates the difference in cell size betweencancer and normal cells.

FIG. 11 illustrates the separation of cancerous and benign breast cellsby use of a diagnostic device as described herein.

FIG. 12 illustrates the displacement of two different cell types on adiagnostic device as described herein.

FIG. 13 illustrates diagnostic devices as described herein during use.

FIG. 14 compares the volume of water transported through variousembodiments of a materials transport devices as described herein.

FIG. 15 illustrates the separation of normal and cancer cells by use ofa diagnostic device as described herein.

FIG. 16 presents the differences in transport capabilities for devicesas disclosed herein incorporating channeled fibers as compared to roundfibers. Results include the difference in wicking rate of constructs(FIG. 16A), the transport of cells to an upper region of devices (FIG.16B) and the infiltration of cells into a scaffold incorporated withdevices (FIG. 16C).

FIG. 17 presents the differences in transport capabilities for devicesas disclosed herein including alginate absorbent caps on one end ascompared to devices without the absorbent caps. Results include thedifference in volume of absorbed fluid over time (FIG. 17A) and thepercentage of cells isolated in the alginate-capped constructs (FIG.17B).

DETAILED DESCRIPTION

Reference will now be made in detail to various embodiments of thedisclosed subject matter, one or more examples of which are set forthbelow. Each embodiment is provided by way of explanation of the subjectmatter, not limitation thereof. In fact, it will be apparent to thoseskilled in the art that various modifications and variations may be madeto the disclosed subject matter without departing from the scope orspirit of the disclosure. For instance, features illustrated ordescribed as part of one embodiment may be used with another embodimentto yield a still further embodiment.

In general, the present disclosure is directed to devices that cantransport biological material. More specifically, disclosed devicesincorporate capillary channeled fibers that can effectively transportliving cells as well as other biological materials such as nutrients,growth factors, waste materials, etc.

Beneficially, it has been discovered that capillary channeled fibers candifferentially transport different cell types, particularly when thefibers are held in a vertical orientation. This has led to thedevelopment of diagnostic devices that can be utilized to separate celltypes from one another. For instance, the diagnostic devices can isolatecancer cells from healthy cells in a heterogeneous cellular solution.Moreover, in addition to separating cells of different types, cells thatcan be separated from one another by use of the diagnostic devices caninclude similar cell types at a different stage of disease ordevelopment as well as polynucleic acids (DNA and RNA) from othermaterials. For instance, cancerous cells can be separated from healthycells of the same type (e.g., cancerous breast cancer cells can beseparated from healthy breast epithelial cells) as well as from othercancer cells of the same type that have varying metastatic capabilities,and progenitor cells can be separated from further differentiated cells(e.g., mesenchymal stem cells can be separated from macrophage cells).

According to one embodiment, the capillary channeled fibers can becombined with a sorptive material that can function as a pump toencourage flow of biological materials through the fibers and providetemporal/spatial transport of cells or other biomolecules. While thiselement can be of great benefit in the diagnostic devices, it has alsoprovided a route to formation of tissue engineering scaffolds that canmore effectively transport biological materials into and out of thescaffolds and the associated developing cellular construct. Withoutwishing to be bound to any particular theory, it is believed that theimproved capillary action provided by the sorptive material of thedevice can improve transport of materials (e.g., cells, growth factors,etc.) into the interior of the scaffold and can also provide for themovement of materials to and from the cells following location of thecells in the interior of the scaffold. The construct can also improvethe initial cell seeding and infiltration of a scaffold by distributioncells homogeneously. The improved transport properties throughout thescaffold can provide for viable cells throughout a cellular construct.Moreover, the improved viability of the cells within the construct canbe maintained without the necessity of perfusion under pressure of theculture media. Accordingly, the cellular construct can better maintainviability, for instance following removal from an in vitro environmentto an in vivo environment, i.e., following implant.

Capillary channeled fibers as may be utilized in the devices can includeany fiber that incorporates one or more open channels along the axiallength of the fiber. Thus, the capillary channeled fibers may also bereferred to as lobed fibers, channeled fibers of non-circular crosssection, or the like. For example, and with reference to FIG. 1,channeled fibers 20 define a non-circular cross section and include aplurality of co-linear channels 22 extending along the axial length ofthe exterior surface of the fiber 20. Each channel 22 is defined by apair of opposed walls 23 that extend longitudinally along a length ofthe fiber and form part of the exterior surface of the fiber 20. In oneembodiment, these channels 22 and walls 23 extend down the entire lengthof the fiber 20 parallel to the longitudinal axis of the fiber 20 andare co-linear on each fiber 20. Examples of suitable capillary-channeledfibers have been described in U.S. Pat. No. 5,200,248 to Thompson, etal., and U.S. Pat. No. 5,972,505 to Phillips, et al., the entirety ofeach of which is incorporated herein by reference.

The channels of a fiber 20 can be designed so as to encourage flow therethrough via capillary action. For instance, a capillary channeled fiber20 can have channel wall thicknesses of less than about 50 μm, less thanabout 10 μm, or less than about 5 μm, in one embodiment. The width of anindividual channel can generally be less than about 0.5 mm, for instanceless than about 0.3 mm, or less than about 0.1 mm. Channel widths fromabout 5 μm to about 0.5 mm can be used, for instance from about 30 μm toabout 100 μm. The individual channels of a fiber 20 can generally bebetween about 15 μm and about 50 μm in depth, i.e., the height of thewalls defining a channel there between. The capillary channels can be ofany suitable shape. For instance a channel can define a regularcross-sectional shape (e.g., U-shaped, V-shaped, etc.), or can beirregular in cross-sectional shape. Generally, the capillary channels ofa fiber can be of regular cross-sectional shape, with capillary channelwalls that are substantially parallel to one another in cross-section.The capillary channel walls can be substantially perpendicular to thestraight chords closing the capillary channels to which the wall servesas a boundary, though this is not required.

In one embodiment, a capillary channeled fiber 20 can satisfy theequation:(1−X cos(θ_(a))<0,

-   -   wherein        -   θ_(a) is the advancing contact angle of water measured on a            flat film of the same material make-up as the fiber, X is a            shape factor of the fiber cross-section that ranges from            about 1.2 to about 5 and satisfies the equation

$X = \frac{P_{W}}{{4r} + {\left( {\pi - 2} \right)D}}$

-   -   wherein    -   P_(w) is the wetted perimeter of the fiber (i.e., the complete        perimeter of the entire fiber cross section)    -   r is the radius of the fiber cross section (the radius of the        circle circumscribing the entire fiber cross-section, and    -   D is the minor axis dimension across the fiber cross section.

In another embodiment, a fiber can satisfy the equation(1−X cos(θ_(a)))<−0.7.

In general, the capillary channeled fibers can be polymeric, though thisis not a requirement of the fibers. When considering polymeric capillarychanneled fibers, a polymer forming the fiber can generally be of anamorphous or semicrystalline character. Particular polymers as may forma capillary channeled fiber 20 can include degradable or non-degradablebiocompatible polymers, depending upon the desired application of thedevice. For instance, if the capillary channeled fibers are a componentof an implantable tissue engineering scaffold, it may be desirable toutilize degradable biocompatible fibers.

Examples of suitable polymers include, but are not limited to,polyesters, polylactides, poly-®-hydroxybutyrates, polycaprolactones,polyglycolides, polyetheresters, rayon, acetate, polyamides (e.g.,nylon), polyolefins, polyacrylates, polydioxanones, polytrimethylenecarbonates, polyanhydrides, polycarbonates, polyoxyalkylene ether,polyurethanes, alkylene oxide compounds, polysaccharides,polyphosphazenes, polyethylene oxide-polypropylene glycol blockcopolymers, fibrin, polyvinyl pyrrolidines, hyaluronic acid, collagen,chitosan, polyvinyl alcohol, copolymers and blends of such polymers, andthe like.

Capillary channeled fibers as may be incorporated in a device areavailable in the retail market and include fibers available from, butnot limited to, COOLMAX® and ANTRON® fibers, which are manufactured byInvista of Waynesboro, Va.; HIGHLIGHTS™ fibers, which are manufacturedby Superior Threads of St, George, Utah; and 4DG™ fibers, available fromFiber Innovation Technology of Johnson City, Tenn.,

Different fabrication approaches can be utilized to form channeledpolymer fibers 20 of the devices. For instance, extrusion processes asare generally known in the art such as melt spinning, wet spinning, anddry spinning can be used. Melt spinning as well as other spinningmethods involve the use of an extrusion die (sometimes referred to as aspinneret) which has an orifice design which roughly corresponds to thecross-sectional shape of the capillary channeled fiber immediately uponexit from the die. Melt spinning involves melting the polymer (or apolymeric composition), applying pressure to extrude the melt through anextrusion die, and cooling and drawing the extruded structure to thedesired size. Depending upon orifice design, processing conditions,polymer composition, and other factors known to those skilled in theart, the final cross-sectional shape of a fiber can deviate from theorifice design somewhat. The extrusion die orifice design, the polymercomposition, the temperature at which it is molten, and processingconditions of the extruded composition during drawing and cooling can beof importance in forming a capillary channeled fiber 20.

A method can include forming polymeric capillary channeled fibers fromamorphous or semicrystalline polymers that can resist breakage upondrawing over a relatively large temperature range, particularly astemperature approaches the glass transition temperature. Whereas suchpolymers are commonly extruded at relatively high temperatures, aformation method can include operating the extruder at temperaturescloser to the glass transition point to reduce viscosity of the melt andconsequently facilitate higher polymer throughput through the extrusiondie and obtain improved shape retention of the capillary channelstructures relative to the extrusion die orifice design. However, as isknown in the art, the optimal temperatures for extrusion will vary frompolymer to polymer. Following extrusion, the nascent fiber can be cooledrelatively rapidly during a drawing process for shape retention.

In one embodiment, two or more capillary channeled fibers 20 can becombined together to form a bundle, as is illustrated in FIG. 1. As canbe seen, when bundled together, the bundle includes the channels of eachfiber as well as one or more closed channels 21 formed between the twoadjacent fibers. Closed channel 21 can function as a conduit to carrymaterials through a device in a semi-isolated state. For instance,channel 21 can function as a closed channel, but can still maintaincommunication and interaction with the surroundings due to the junctionsformed between the adjacent fibers. Thus, a channel 21 formed betweenadjacent fibers can provide both the fluid transport properties of aclosed channel and the interaction with the surrounding environment ofan open channel. These inter-fiber spaces can enhance the wicking rate.The multiple fibers can be bundled by any of various mechanism, e.g.,twisted, braided, intertwined, knotted, patterned weave, etc. Thespecific pattern of bundling can be designed to vary the inter-fiberspaces as desired, which can be used to adjust the transport propertiesof the bundle of fibers.

Fluid conduits as provided by the bundles of capillary channeled fibers20 can also include additives such as biologically active agents orother materials either within the polymeric structure of the fibers orattached to the surface of the fibers, such as coatings to affect fluidflow. For instance, capillary channeled fibers can include growthfactors, antibiotic agents, and the like incorporated within the fiberstructure that can leach out of the fiber for release or can be releasedas a fiber degrades.

Biologically active agents can be located on the surface of a fiberaccording to any suitable coating or surface application process. Forinstance, biologically active agents can be temporarily or permanentlybonded to a capillary channeled fiber 20. In general, an agent can bebonded to a fiber via an existing chemical functionality of the agent,such as amine, carboxylate, or thiol groups that can allow covalent,non-covalent, charge/charge, or any other type of bonding of the agentto a fiber surface while maintaining the desired activity of the agent.In such an embodiment, the base fiber composition can be selected so asto incorporate an anchoring mechanism for the agent. By way of example,a biologically active agent that includes a protonated aminefunctionality can be bonded to a polymeric fiber surface includingnegative charge groups.

Surface chemistry modification of a fiber can also be carried out toform a coating or alter the surface characteristics of the fibers and/orto encourage bonding of a biologically active agent to the fiber. Fibersurface chemistry modification processes can include, withoutlimitation, alkaline treatments, plasma treatments, and the like. Fibersurface modification can encourage bonding of an active agent to thesurface via any means. For example, fiber surface modification canincrease the number of carboxylate groups on a fiber surface availablefor bonding to a desired active agent.

According to one embodiment, a grafting process may be employed forfunctionalizing a fiber surface with a desired chemistry. For instance,an at least bifunctional polymer possessing desired reactive functionalgroups such as carboxy, anhydride, amino or hydroxy groups may be firstgrafted to a fiber surface utilizing a portion of the functional groupsof the polymer. Remaining functional groups of the polymer may then beutilized to attach additional functional materials to the fiber surface,e.g., biomolecules, micro-particles, nano-particles, and the like. Forexample, a poly(ethylene terephthalate) (PET) fiber can be modified toinclude a polyacrylic acid layer, with further functionalization asdesired to incorporate specific biologically active agents at the fibersurface. Surface modification of substrates in accordance with thisgrafting process is taught by U.S. Pat. No. 7,026,014 to Luzinov, etal., the entirety of which is incorporated herein by reference.

Direct surface modifications can also be used to establish a surface towhich active ingredients can be anchored. For example, a polyamide fibersurface can be treated with ethylene-diamine to form a surface that isrich with both carboxylate and amine functionalities that can then beutilized to bond specific biologically active agents.

A plurality of fibers can be combined to form a bundle according to anydesired method. For instance, a bundle of fibers can include 2 or morefibers, for instance from about 2 to about 10 fibers, or from about 3 toabout 7 fibers can be combined to form a bundle. Fibers can be combinedthrough simply mechanical means, for instance by twisting, braiding,knotting, intertwining, weaving, etc. a plurality of fibers together, orthe fibers can be adhered together, for instance under pressure and heator by use of an adhesive to form spot bonds between the fibers,provided, however, that any bonding process does not deform thecapillary channels of the fibers and prevent or interfere with flowthrough the channels.

The materials transport devices can be diagnostic devices that utilizethe capability of the capillary channels of the fibers to differentiallytransport cells in a test sample. FIG. 2 schematically illustrates abundle 310 of capillary channeled fibers as may be incorporated in adevice and includes FIG. 2A, which illustrates a complete fiber bundleand FIG. 2B, which illustrates the fiber bundle split into a top regionand a bottom region. During use, the bundle 310 can be located in avertical orientation in a test sample that includes a first cell type301 and a second cell type 302 with a first end 311 of the bundle 310 inthe test sample. The transport properties of the device can cause thecells 301, 302 to be wicked up the channels of the bundle 310. Followinga period of time for wicking, generally about 15 minutes or more, forinstance between about 15 minutes and 1 hour for a fiber or fiber bundleof a height of about 5 cm (for instance between about 2 and about 10cm), the cells within the sample will preferentially rise to a heightwithin the bundle 310 depending upon the cell type. For instance, asshown, the cells 301 can preferentially remain in the bottom region ofthe bundle 310 and the cells 302 can preferentially rise to the topregion of the bundle 310. It should be understood that a device can beutilized to separate multiple cell types, and is not limited toseparating only two cell types from one another,

Without wishing to be bound to any particular theory, it is believedthat the mechanism for cell separation within the channels of the fibersor fiber bundles is due to the physical and functional properties of thecells, wicking characteristics of the fibers, and the cell-fiberinteraction. Physical properties that may play a role include cell size,deformability, surface friction or charge, and expression of celladhesion molecules. For instance, the size and shape of the cells mayplay a role in the ability for cells to penetrate smaller channelscreated from the bundling of the fibers,

Fiber bundle architecture can affect the wicking rate and verticaldisplacement of different cell types. For example, cross-sectionalimages of wicking fiber bundles depict channel and inter-fiber spacessizes ranging from 5 μm to 95 μm. The size of the individual fibers, thetension of a bundle, the hydrophobicity, and the inter-fiber space canall play a role in the wicking of liquid and cell-interaction.

Other cellular properties that are believed to influence verticalmovement of cells within a channel include cell membrane impedance,expression of adhesion molecules, and cells stiffness. As is known,membrane impedance, cell dielectric properties and cell electricproperties vary between cell types. Electrical properties of the cellcan influence the cell-interaction with the fiber and the resultantvertical movement. Cells with greater impedance or surface charge mayhave more interactions with a fiber, hindering the vertical cellmovement. By way of example, it has been found differences in cellularimpedance of cancer cells of varying aggressiveness. Specifically, ithas been found that a more aggressive cancer cell type can have asignificant reduction in impedance as compared to a less aggressivecancer cell. Additionally a correlation has been found betweenmetastatic progression and membrane impedance reduction. Cell-fiberinteraction and overall cell displacement of cell types can thus beaffected by different membrane impedance of the different cell types.

Expression of cell adhesion molecules can also play a role in thecell-fiber interaction and ability of a cell to move through a confinedspace. Metastatic cells have been shown to lose endothelial adhesionmolecules and transition into a more motile mesenchymal phenotype. Forinstance, cancerous cells with fewer adhesion molecules may have lesssurface friction and altered shape and can thus have less interactionwith the fiber allowing the cells to wick greater distances.

Cell deformability can influence the ability of cells to deform,penetrate, and wick vertically through the channels of a fiber or fiberbundle. In general, research has shown that cancer cells have asignificantly lower young's modulus compared to normal cells.Researchers have found a correlation between increased metastaticpotential of cancer cells and higher deformability. Thus, cancer cellsmay be much softer and deform easier, allowing them to migrate morereadily.

Knowledge with regard to differences between cell types to be separated(e.g., difference in stiffness, morphology, size, membrane impedance,etc.) can be used to design a device for optimal separation of celltypes. For instance, given a known difference in size between celltypes, the fibers of the device can be formed with particular channelsizes so as to better exclude or slow the passage of the larger celltype. Similarly, the stiffness or shape of the fibers and/or channelscan be designed to optimize separation of particular cell types havingknown differences in characters. In one embodiment, the fibers can besurface functionalized to interact more strongly with one cell type thananother based on differences in adhesion molecules or other cell surfacereceptors on the different cells. In addition, the hydrophobicity of thefibers can be varied, which can alter cell interaction and wickingbehavior of the cells along the fiber channels.

FIG. 3 illustrates one embodiment of a materials transport device thatincorporates a capillary channeled fiber or fiber bundle. As can beseen, the device of FIG. 3 is a vertical test system that can be used toanalyze the vertical movement of a mixture of different cells 401, 402along the fiber or fiber bundle 410.

The device includes a well 420 and a brace 430 for holding the fiber orfiber bundle 410 in a vertical orientation within the well. The brace430 can be any structure that can hold the fiber or fiber bundle 410 ina vertical orientation with a first end of the fiber or fiber bundle 410within the well 420. For instance, a brace 430 can include a supportingtoroid of a size to hold the fiber or fiber bundle 410 securely in avertical orientation. The brace 430 can be attached to the well 420 orfree-standing, as desired. In another embodiment, the brace 430 can be acomponent of a lid for a well 420. For instance, a lid can include anaperture therethrough from the bottom side of the lid to the top side ofthe lid. The aperture can function as the brace 430 and can hold thefiber or fiber bundle 410 in a vertical orientation after the lid hasbeen placed on the well 420.

During use, the fiber or fiber bundle 410 can be located with one end inthe bottom of the well 420 such that the end is immersed in a mixturethat includes the cell types 401, 402 to be separated. The fiber orfiber bundle 410 can be braced in a vertical orientation by use of thebrace 430 and following a period of time the cells 401, 402 candifferentially wick to different heights along the fiber or fiber bundle410. Following the contact period, the cells can be removed from thefiber or fiber bundle 410 for further examination. For instance, thefiber or fiber bundle can be cut into separate regions (e.g., a topregion and a bottom region) and the cells adhered to each region can berinsed from the regions and further examined, as desired.

In one embodiment the capillary channeled fibers can be combined with asorptive material that can improve the wicking capability of the fibers.For instance, FIG. 5 and FIG. 6 illustrate bundles 510, 610 that includea plurality of capillary channeled fibers. At the end 512, 612 of eachrespective bundle 510, 610 is a cap 514, 614 that encloses one end ofthe bundle 510, 610. The cap includes a sorptive material that canencourage wicking through the fibers and further improve the transportcapabilities of the device.

The sorptive material of the cap can include any sorptive hydrophilicmaterial as is known in the art including both absorbent materials andadsorbent materials. The sorbent material can thus include any one of orcombination of absorbent materials and/or elements and adsorbentmaterials and/or elements. Sorbent materials that may be used includefabrics, foams, fibrous structures and the like. Suitable materialsinclude as non-limiting examples natural and synthetic polymericabsorbents, super-absorbents and celluloses. Fibrous absorbentsmanufactured from absorbent fibers such as alginate fibers and sodiumcarboxymethyl cellulose fibers, otherwise referred to as hydrofibers,are encompassed herein.

In general, absorbent materials are those that can sorb fluid through aprocess of absorption, similar to a sponge, in which a liquid diffusesinto the volume and/or structure of the absorbent and becomes a part ofthat volume and/or structure. For example, the sorbent can pick up andretain a liquid distributed throughout its molecular structure causingthe absorbent to swell. The liquid can cause the structure to swellabout 50% or more, in one embodiment.

Certain exemplary embodiments of absorbents include, but are not limitedto, comminuted wood pulp fluff, cellulose fibers, polymeric gellingagents, hydrophilic nonwovens, cellulose, sodium polyacrylate, cotton,polyethylene terephthalate, polyethylene, polypropylene, polyvinylchloride, ABS, polyamide, polystyrene, polyvinyl alcohol, polycarbonate,ethylene methacrylate copolymer, polyacetal, etc., and combinations ofadsorbent materials.

Adsorbents, on the other hand, remove fluid through a process ofadsorption by retaining a liquid on their surface including pores andcapillaries. Liquid accumulates on the surface of an adsorbent byforming a film of molecules or atoms that are retained thereon as aconsequence of surface energy. In some embodiments, an adsorbentmaterial can include one or more insoluble materials (or at leastpartially insoluble) that can be coated by a liquid on their surface.For example, the adsorbent can be a structure formed from insolublefibers. The structure can be porous, as voids or spaces can be locatedbetween the individual fibers. Thus, liquid can accumulate on thesurface of the fibers, thereby filling the voids between the fibers.Typical adsorbents will adsorb fluid without swelling more than about50% in excess liquid.

Certain exemplary adsorbent materials include, but are not limited to,oxygen-containing compounds, carbon-based compounds, and/or polymerbased compounds, among others. For example, adsorbent materials caninclude silica gels, alumina, zeolites, activated carbon, graphite,cellulose, porous polymer matrices, perlite, metal hydroxides, metaloxide filled cellulose acetate, butyrate and -nitrate, polyamide,polysulfone, vinyl polymers, polyesters, polyolefins and PTFE,polyurethane, as well as porous glass or glass ceramics, graphite oxide,polyelectrolyte complexes, etc., and combinations of adsorbentmaterials.

According to one embodiment, the sorptive material can be a hydrogel.Hydrogels are water-sorbable and generally cross-linked polymericstructures, usually having low modulus and compressive strength.Collagen is known to be an excellent hydrogel matrix material and hasbeen found in diverse applications in regenerative medicine due to itsbiocompatibility and biodegradability. Alginate, a polysaccharideextracted from seaweed, has been used widely in tissue engineeringapplications due to its ability to gently gel in the presence ofdivalent ions such as calcium chloride.

According to another embodiment, a sorptive material can include analginate sponge. Alginate sponges are highly porous, lyophilizedsubstances with a lengthy history of use in connection with cellculture. The alginate of a sponge generally refers topharmaceutical-grade plant-derived alginate. Pore size and shape isgenerally homogenous, with pores ranging from 50 to 200 μm in size. Inone embodiment, the pores may be interconnecting. Alginate-based fibrousabsorbents such as are used in dressings containing fibers of calciumalginate, or blends of sodium and calcium alginate, or fibers of sodiumcarboxymethyl cellulose can be utilized as a sorptive material.

The sorptive material can be further treated to provide additionalfunctionality to the device. For instance, a sorptive material, such asa sorptive polymer can incorporate functional groups that can bind tocells and/or biomolecules to be transported by the device. In oneembodiment the sorptive material can include growth factors orbiomolecules to enhance cell development and/or to deliver biomoleculesto an area in the scaffold.

The sorptive material can be altered by modifying the cross-linking, theamount enclosing the fibers, the type of sorptive material etc. Suchmodifications can change the displacement of cells and which cell typesare collected,

In one embodiment, the cap can be coated, for instance with ahydrophobic or water impermeable material that can isolate the interiorof the cap and prevent Materials that collect in the cap from escape aswell as to prevent sorption of materials in the local environment by thesorptive material of the cap. For example, the cap can be coated, forinstance with a hydrophobic or liquid impermeable coating, to alter thedegradation characteristics of the sorptive material and prevent fluidsorption from surrounding tissue. Coating materials can include, forexample and without limitation, cyanoacrylates, agarose, polyethyleneglycol, etc.

The cap including sorptive material can be attached to one end of acapillary channeled fiber or bundle of fibers according to any suitablemeans. For instance, the sorptive material can adhere to the end of afiber or bundle through charge/charge interaction, through covalent orionic bonding, by use of a biocompatible adhesive, and so forth.

The cap can cover one end of the fibers such that the fiber ends areenclosed within the cap. The cap can generally enclose a portion of thefiber or fiber bundle, for instance about less than about half of thefiber or fiber bundle, less than about one third of the fiber or fiberbundle, less than about 20%, less than about 10%, or less than about 5%of the fiber or bundle, in some embodiments.

FIG. 4 illustrates a cross sectional view of a sorptive cap 116 thatencapsulates a bundle 114 of capillary channeled fibers 120. A cap 116including the sorptive material can enclose the ends of the fibers 120such that the channels 121 between fibers and the channels 122 along thefibers terminate within the cap 116. The sorptive qualities of the cap116 can encourage wicking through the channels 121, 122 and improvematerials transport through the fibers.

Diagnostic systems can be designed that incorporate the materialstransport devices. As shown in FIG. 6, a diagnostic system including awell 620 and a brace 630 (in this embodiment a lid with an aperturetherethrough) can utilize materials transport device that includes afiber bundle 610 with a cap 614 enclosing one end 612 of the fiberbundle 610. The cap 614 can enclose the end of the fiber bundle 610 andencourage wicking through the channels of the capillary channeled fibersof the bundle 610.

The device includes a bundle 610 of cell- and liquid-wicking fibers ofnon-circular cross-section and a cap 614 that includes a biocompatiblesorptive material, such as alginate. The cap 614 interfaces only one endof the bundle 610 as described earlier. The sorptive material andwicking fiber device can contact cells or tissue sample from a subject.Different cell types can be transported through the fibers and collectin the cap 614 at different rates. Thus, cells can be isolated anddistinguished based on their vertical distance traveled in the fiber andcollection in the cap 614. The device can be used as a diagnostic deviceby collecting or isolating cells in the cap 614 and/or the fiber bundle610.

In one embodiment, the cap 614 can be removable. Thus, in thisembodiment, following a period of wicking, the cap 614 can be removedand replaced with another cap that includes the same or differentsorptive materials, and the second cap can collect different cell typesmoving at different rates,

The materials transport devices can also be of benefit in tissueengineering applications. For example, a device that includes a bundleof capillary channeled fibers and a cap with a sorptive material can beincorporated in a tissue engineering scaffold matrix so as to improvetransport of cells, nutrients, wastes, etc, throughout the scaffold.Accordingly, as a cellular construct develops on the scaffold, inclusionof one or more materials transport devices can not only encourageinitial development of a large cellular construct, but can also preventnecrosis of new cells within the center of the developing tissueconstruct.

The device can transport fluids, biomolecules, and cells temporallyand/or spatially to targeted locations within a tissue engineeringscaffold. This device can be incorporated into a scaffold in variousorientations to direct the movement of cell and biomolecules throughoutthe scaffold. For example, in one embodiment, the cap of the device canbe located within the interior of the scaffold matrix and the second endof the bundle can be located outside of the scaffold matrix, forinstance to target specific vascular or progenitor cell sources in theimplant site. In this embodiment, the cap of the device can function asa passive pump and can encourage the flow of cells, nutrients, andbiomolecules from the exterior are (e.g., surrounding tissue of animplanted scaffold) to be directed to the interior of the scaffold.

In one embodiment, the device can be incorporated into a scaffold matrixwith the sorptive material cap on the exterior of the matrix and thebulk of the fiber bundle on the interior of the scaffold matrix. In thisembodiment, the materials transport device can encourage the flow ofwastes and harmful byproducts from the center of the scaffold to theexterior of the matrix (e.g., the surrounding implant site).Accordingly, the capillary channeled fibers can provide channels for thewastes to travel and the cap can function as a passive pump and in oneembodiment can collect waste products within the cap.

One or more material transport devices can be incorporated within tissueengineering scaffolding matrices as are generally known in the art. Inone embodiment; the overall diameter of a scaffolding matrix can berelatively large; for instance greater than about 10 mm, or greater thanabout 15 mm. For example, an engineering scaffold including a porousmatrix and one or more material transport devices can be incorporatedtherein can be between about 10 and about 30 mm in cross sectionaldiameter, or about 20 mm in diameter. As utilized herein, the crosssectional diameter of a scaffold refers to the diameter of a circle thatencloses a cross section of the scaffold. Thus, the tissue engineeringscaffolds are not limited to any particular shape or geometry.

Fiber bundles of different devices within a scaffold can run generallyparallel to one another or can run at angles to one another. In oneembodiment, the fiber bundles of multiple devices can run generallyparallel to one another, which can increase the mechanical integrity ofthe scaffold and the cellular construct that is developed on thescaffold. In one embodiment, the fiber bundles can be oriented basedupon sources of vascular/growth factor/cell supply at the implant site.

The matrix of a tissue engineering scaffold can generally be a porousmatrix that can encourage the growth and development of tissue thereon.In one particular embodiment, the matrix can be a polymeric matrix.Polymers of the matrix can include any biocompatible polymer and, in oneembodiment, can include implantable polymers. In general, the preferredmaterial of the matrix can depend upon the intended use of the scaffold.For example, in those embodiments in which a tissue construct developedon/in the scaffold is intended for implantation, the matrix can bedeveloped so as to anticipate the implantation environment.

In general, any synthetic or natural polymer approved for human clinicaluse can be utilized in forming the porous matrix. For instance, polymersutilized in medical and pharmaceutical applications such as surgicalsuture materials or in controlled release devices can be utilized. Byway of example, a scaffold matrix can be formed of one or more polymersincluding, without limitation, polyesters, polyanhydrides;polyorthoesters; polyphosphazenes; polyhydroxy acids such as polylactide(PL), polylactic acid (PL), polyglycolide (PG), polyglycolic acid (PGA);and polycaprolactone (PCL); aliphatic polyesters; poly(amino acids);polyalkylene oxalates; polyamides, poly(iminocarbonates); polyoxaesters;polyamidoesters; polyarylates; polyhydroxyalkanoates; peptides andpolysaccharides such as agarose; dextran; hyaluronic acid; chitin;heparin; collagen; elastin; keratin; albumin; polymers and copolymers oflactide; glycolic acid; carboxymethyl cellulose; polyacrylates;polymethacrylates; epoxides; silicones; polyols such as polypropyleneglycol; polyvinyl alcohol and polyethylene glycol and their derivatives;alginates such as sodium alginate or crosslinked alginate gum;polycaprolactone; polyanhydride; pectin; gelatin; crosslinked proteins;and the like.

In one embodiment, a polyester such as a copolymer of polylactide can beutilized. For instance copolymers of polylactide and polycaprolactone(e.g., ∈-polycaprolactone), polyglycolic acid, trimethylene carbonate,or p-dioxanone can be utilized in forming a scaffold matrix. Thesecopolymers are biocompatible and bioresorbable in that their degradationproducts are low molecular weight compounds such as lactide and glycolicacid that can enter into normal metabolic pathways. Furthermore,copolymers of polylactide can offer the advantage of a large spectrum ofdegradation rates from a few days to years by simply varying thecopolymer ratio of lactide to glycolic acid.

Polymers of a matrix can include lactide polymers such aspoly(L-lactide) (PLL), poly(DL-lactide) (PDL), and copolymers thereofincluding poly(lactide-co-caprolactone) (PL/PCL). The co-monomer(lactide:caprolactone) ratios of a PL/PCL copolymer can generally bebetween about 100:0 and about 50:50. For example, the co-monomer ratioscan be between about 85:15 and about 50:50. Blends of PL with PCL canalso be utilized, for instance a PLL:PCL blend at a ratio between about85:15 and 50:50 can be utilized.

A matrix can be formed of a biocompatible hydrogel and in oneembodiment, an implantable hydrogel. For instance, a matrix can beformed of a biodegradable hydrogel. Suitable polymers of a matrix caninclude non-crosslinked and crosslinked polymers. A matrix including acrosslinked polymer can optionally include hydrolyzable portions, suchthat the portion can be degradable following implant of a tissueconstruct. For example, a hydrogel matrix can include a hydrolyzablecomponent, such as polylactide. The crosslink density can be designedaccording to standard methods as are generally known in the art tocontrol the rate of degradation of the matrix following implant,

A matrix can be formed according to any method as is generally known inthe art. For instance, a matrix can self-assemble upon mere contact ofthe various components or upon contact in conjunction with the presenceof particular external conditions (such as temperature or pH).Alternatively, assembly can be induced according to any known methodfollowing mixing of the components. For example, step-wise or chainpolymerization of multifunctional monomers or macromers can be inducedvia photopolymerization, temperature dependent polymerization, and/orchemically activated polymerization. Optionally, a matrix can bepolymerized in the presence of an initiator. For example, in oneembodiment, a matrix can be photopolymerized in the presence of asuitable initiator such as Irgacure® or Darocur® photoinitiatorsavailable from Ciba Specialty Chemicals. In another embodiment, acationic initiator can be present. For example, a polyvalent elementalcation such as Ca²⁺, Mg²⁺, Al³⁺, La³⁺, or Mn²⁺ can be used. In anotherembodiment, a polycationic polypeptide such as polylysine orpolyarginine can be utilized as an initiator.

A scaffold matrix can include one or more additives. For example, in oneembodiment, a tissue engineering scaffold can be utilized for thedevelopment of an osteogenic tissue construct, and ceramic additives canbe included in the matrix. Polymer/ceramic composites can provide thestructural stability and controlled degradation rate of implantablepolymers in conjunction with improved osteogenesis through inclusion ofa calcium phosphate mineral phase in the portion. For instance,polymer/ceramic composites as disclosed by Laurencin, et al. (U.S. Pat.No. 5,626,861), Laurencin, et al, (U.S. Pat. No. 5,866,155),andArmstrong, et al. (U.S. Pat. No. 6,417,247), all of which areincorporated herein by reference, can be utilized in a tissueengineering scaffold.

Bioactive agents as may be incorporated in a tissue engineeringscaffold, either as a component of the matrix, as a component of amaterials transport device (e.g., the capillary channeled fibers), orboth can include growth factors (e.g., Transforming Growth Factor-beta(TGF-®)), nutrients and the like to encourage growth and development ofa tissue construct on the scaffold. For example, the tissue engineeringscaffold can include hormones, analgesics, anti-inflammatory agents,chemotherapeutic agents, anti-rejection agents, proteins and peptides(e.g., RGD peptides), polysaccharides, nucleic acids, lipids, andnon-protein organic and inorganic compounds. Additives can include thosethat can exhibit biological effects such as osteogenic additives,osteoinductive additives, osteoconductive additives, growth factors,differentiation factors, steroid hormones, cytokines, lymphokines,antibiotics, and angiogenesis promoting or inhibiting factors and soforth.

To promote cell attachment, cell adhesion factors such as laminin,pronectin, or fibronectin or fragments thereof, e.g.arginine-glycine-aspartate, may be coated on or otherwise incorporatedon or in a scaffold. A scaffold and/or the transport device may also becoated or have incorporated therein cytokines or other releasable cellstimulating factors such as; basic fibroblast growth factor (bFGF),transforming growth factor beta (TGF-®), nerve growth factor (NGF),growth factor-1 (IGF-1), growth hormone (GH), multiplication stimulatingactivity (MSA), cartilage derived factor (CDF), bone morphogenicproteins (BMPs) or other osteogenic factors, anti-angiogenesis factors(angiostatin), vascular endothelial growth factor, platelet-derivedgrowth factor and insulin-derived growth factors (IGF).

DNA such as a gene sequence or portion thereof, coding for a growthfactor or other of the auxiliary factors mentioned above may also beincorporated into a scaffold. The DNA sequence may be “naked” or presentin a vector or otherwise encapsulated or protected. The DNA sequence mayalso represent an antisense sequence of a gene or portion thereof.

Additives can also include tracking or monitoring agents such as,without limitation, radiopaque materials such as barium, or otherimaging agents.

It should be understood that any additive is not limited to any specificportion of the scaffolds. Additives may be incorporated in any suitableportion of a scaffold, as is known to one of ordinary skill in the artincluding one or more capillary channeled fibers, one or more fiberbundles, and/or any portion of a matrix.

The porosity of a matrix can be formed according to any methodology. Ingeneral, the porous matrix can define a porosity of between about 10 andabout 90 volume %, for instance between about 20 and about 50 volume %,and a pore size of between about 30 μm and about 300 μm, for instancebetween about 50 μm and about 250 μm, or between about 100 μm and about200 μm.

According to one embodiment, the tissue engineering scaffold can beformed according to a particulate leaching method. FIG. 7 schematicallyillustrates one such method. As can be seen, a matrix can be formed bycombining a mixture 302 including a polymer in a solvent with asacrificial porogen 314 that will later be removed to provide thedesired porosity. The composite mixture 304 thus formed is molded arounda plurality of materials transport devices 311 held in a mold 306 andthe solvent allowed to evaporate. Individual transport devices 311 canhave a distance between one another to encourage transport of materialsthrough the porous matrix. For instance, the individual transportdevices 311 can be between about 1 mm and about 10 mm apart (e.g.,center-to-center of the bundle spacing), or between about 3 and about 7mm apart, in one embodiment.

The resulting mold 308 can then be heated slightly beyond the T_(g) forthe polymer of the matrix to ensure complete bonding of the polymer.Once cooled, the mold 308 can be placed in a solvent 312 such that thesacrificial porogen particles are dissolved or leached out to providethe tissue engineering scaffold 310. The porogen can be any suitablesacrificial material, such as a salt, a sacrificial polymericmicrosphere, or the like.

FIG. 8 illustrates another embodiment of a tissue engineering scaffoldincluding a porous matrix 810, a first materials transport device thatincludes a first fiber bundle 812 and a first cap 814 on the first fiberbundle 812 and a second materials transport device that includes asecond fiber bundle 817 and a second cap 818 on the second fiber bundle.In this particular embodiment, the two transport devices are attached toone another such that one end of the second fiber bundle 817 is enclosedwithin the cap 814 of the first device. This may prove helpful inencouraging transport of materials throughout the device, but is not arequirement of a scaffold. In other embodiments, multiple devices of ascaffold can be separated from one another, with a portion of thedevices having the cap section within the scaffold and another portionof the devices having the cap section exterior to the scaffold. Inanother embodiment, the bundle of fibers can be directed toward thesource or sources of the implant site and the absorbent material can belocated within in the scaffold.

During use materials 815 including in one embodiment living cells can betransported into the scaffold in order to form a tissue engineeredconstruct on/within the scaffold. Such cells include autograft cellswhich are derived from a patient's tissue and have (optionally) beenexpanded in number by culturing ex vivo for a period of time beforebeing introduced onto a scaffold. Cell types can include cell types fromany species. By way of example, human cell types as may be loaded onto ascaffold can include, without limitation, stem cells, diseased cells,myocytes, myoblasts, osteocytes, osteoblasts, epithelial cells, andcombinations of multiple cell types.

Cells can be loaded onto a scaffold at any time during use, with thepreferred loading timing/methodology generally depending upon the enduse of the scaffold/construct. For instance, when considering an invitro utilization, in which the tissue engineering scaffold may beutilized for study and/or development, but not necessarily for implant,the cells can be loaded following formation of the tissue engineeringscaffold or even in conjunction with formation of the scaffold, forinstance in conjunction with the formation of the matrix that surroundsone or more materials transport devices. When considering utilization ofa scaffold in an implant application, cells can be loaded on thescaffold prior to implant, for instance at a time prior to implant suchthat a period of growth and development of the cells on the scaffold iscarried out prior to implant. In one embodiment, ex vivo cells of thesubject can be utilized, and the cells can be loaded on the implant atany time prior to implant. For example, ex vivo cells can be loaded ontoa tissue engineering scaffold several hours or days prior to the implantprocedure, so as to provide a time period for the cells to grow anddevelop on the scaffold, or alternatively, cells may be loaded on to thescaffold immediately prior to implant, for instance in the operatingroom prior to implant of the scaffold. In yet another embodiment, atissue engineering scaffold may be implanted in a living subject withoutcells loaded on to the scaffold, and the scaffold may serve as a sitefor natural cell loading, growth, and development for the subject's ownsystem following implant.

The second materials transport device can efficiently transport wastematerials 816 from the interior of the scaffold 810 to the exteriorfield. In one embodiment, the waste materials 816 can be collectedwithin the cap 818, for instance for examination.

The present disclosure may be better understood with reference to theexamples, set forth below.

Example 1

Normal mouse mammary epithelial cell line, NMuMG (ATCC), was stablytransfected with Green Fluorescent Protein (NMuMG-GFP). Cancer mouseepithelial cell line (cells isolated from a mammary tumor thatspontaneously arose in a MMTV-neu transgenic female mouse) was stablytransfected with Red Fluorescent Protein (MMTV-neu-RFP). NMuMG-GFP cellswere cultured in Dulbecco's Modified Eagle's Medium (DMEM, Invitrogen)supplemented with 10% fetal bovine serum (FBS, Gibco), and MEGM singlequots (Lonza) while MMTV-neu-RFP cells were cultured in DMEM(Invitrogen) supplemented with 10% FBS (Gibco), 10,000 U penicillin, and10 mg streptomycin/mL (Sigma-Aldrich). Cells were cultured in a T150flask (Corning) and maintained in a humidified incubator at 37° C. and5% CO₂. Once cells were confluent, NMuMG-GFP at passage 4 andMMTV-neu-RFP at passage 5 were detached using trypsin-EDTA solution(Sigma) and resuspended in growth media to prepare for the verticaltest.

Poly-L-lactide (Natureworks) wicking fiber was extruded withnon-circular cross-sectional dimensions of 0.72 mm×0.55 mm. Each wickingfiber was sliced into multiple individual single wicking fibers of 3.5cm and 10 cm in length. The 10 cm wicking fibers were used to form thewicking fiber bundles. Three of these fibers were twisted using anapparatus at 11 rotations per centimeter twist. The bundle was slicedinto 3.5 cm long sections. Single and bundled wicking fibers werecleaned by soaking in three changes of ethanol for 1 hour, and placedunder ultraviolet light for 6 hours. Samples were then soaked inphosphate-buffered saline (PBS, Invitrogen) solution for 2 hours andair-dried overnight in a sterile hood.

The vertical wicking test of cells utilized a device as illustrated inFIG. 4. The lid contained fitted holes with columns to securely hold the3.5 cm wicking fibers and wicking fiber bundles vertical. Both cancerand normal cell lines were seeded in each well of a low attachment12-well plate at a density of one million cells per well along with 1 mLof growth media. Single wicking fibers or wicking fiber bundles werevertically inserted through the columns so only the bottom 3 mm of thefiber was contacting the cell solution. The set up was placed on aflattop shaker (VWR) at 100 rpm in a humidified incubator at 37° C. and5% CO₂. The vertical displacement of the mouse cancer and normal mammarycells along the wicking were determined at time points of 0.5, 9, and 24hours with the initial time point being fiber placement into the cellsolution.

To assess the cell displacement of both cell types the fibers weretransferred to a well of 6-well plate, rinsed twice withphosphate-buffered saline solution, and fixed for 15 min with 4%paraformaldehyde. After the cells on the wicking fiber constructs werefixed, the fibers were transferred to microscope slides and the verticaldisplacement was evaluated using fluorescent microscopy and imagingsoftware. The entire length of the fiber was imaged using fluorescentmicroscopy using 25× total magnification. FITC was used to view thevertical movement of normal cells transfected with Green FluorescentProtein, and TRITC is used to view the vertical movement of cancer cellstransfected with Red Fluorescent Protein. Imaging software was used todetermine the vertical displacement (μm) of the cells in each imagetaken along the fiber. Images were aligned to qualitatively show thetotal displacement of both normal and cancer cells with total verticaldisplacement quantified by the summation of each individual image.

Images qualitatively showed a vertical separation of cancer cells andnormal cells and depict higher cell densities of cancer cells along thefiber. Imaging software was used to quantify the vertical displacement(μm) of the cells in each image taken along the fiber. The totalvertical displacement was found from the summation of each individualimage along the fiber. Matched pairs test analysis was conducting usingJMP statistical software to compare (p<0.05) the vertical displacementof MMTV-neu-RFP, cancer cells, and MMuMG-GFP, normal cells. The resultsshowed the vertical displacement of MMTV-neu-RFP cells was significantlygreater (p<0.05) than NMuMG-GFP cells after 9 hours and 24 hours (FIG.9). Results suggest wicking fibers can separate cancer cells from amixed cellular solution and potentially isolate the cancer cells fromthe top regions.

Example 2

Mammary epithelial cell line from benign breast tissue, MCF-10A (ATCC),was stably transfected with Green Fluorescent Protein (MCF-10A-GFP).Human breast cancer cell line, MCF-7, (ATCC) was stably transfected withRed Fluorescent Protein (MCF-7-RFP). MCF-10A-GFP cells, passage 5, werecultured in DMEM (Invitrogen) supplemented with 10% FBS (Gibco), 1%Fungizone, and MEGM single quots (Lonza). MCF-7-RFP cells, passage 6,were cultured in DMEM (Invitrogen) supplemented with 10% FBS, 1%Fungizone (Gibco), 10,000 U penicillin, and 10 mg streptomycin/mL(Sigma-Aldrich). Cells were cultured in a T-150 flask (Corning) andmaintained in a humidified incubator at 37° C. and 5% CO₂. Onceconfluent both cell types were removed with trypsin-EDTA solution(Sigma-Aldrich) and resuspended in culture medium to prepare forvertical test.

The average cell size of the MCF-7-RFP and MCF-10A-GFP cells wasmeasured in cell solution. Cells were removed from culture flasks byadding trypsin EDTA solution. After incubation with solution for 15 min,5 million cells were resuspended in 5 mL of growth medium in a 15 mLcentrifuge tube. The centrifuge tube was vortexed to maintain the cellsin solution. A volume of 10 μL of cell solution was added to fivedifferent microscope slides. The MCF-10A-GFP and MCF-7-RFP cells wereimaged with the fluorescence microscope and measured with imagingsoftware. The measurement function of the software was used to determinethe average diameter of each cell line. Results are presented in FIG.10.

A device and method as described in Example 1 was utilized to assess thecell displacement of both cell types. After 24-hours the verticalwicking fiber bundles were removed from the custom 12-well plate and thefibers were sectioned with a blade into a top and bottom region of thefiber. The top and bottom regions were placed in separate wells of a24-well plate. The samples were rinsed with PBS twice and untwistedusing forceps. The cells were removed by adding 500 μL of trypsin-EDTAsolution into the well with the fiber region and placing the plate on aflat-top (VWR) shaker at 200 rpm in a 37° C. incubator. After 15 min thecells were resuspended in 500 μL of growth media and the number ofMCF-10A and MCF-7 in both regions of the fiber were evaluated usingguava easyCyte™ flow cytometry (Guava Technologies). The number ofMCF-10A-GFP and MCF-7-RFP in each region was evaluated by following themanufacturer's instructions for InCyte software (Guava Technologies).Positive and negative controls with known cell densities were used tocalibrate the machine before measurements on the treatments were taken.

The vertical displacement of the human cancerous and benign cells alongthe fiber bundles were determined at time points of 0.25 hours, 2 hours,12 hours and 24 hours with the initial time point being fiber placementinto the cell solution. The matched pairs test analysis in JMP was usedto compare (p<0.05) the amount of MCF-7-RFP and MCF-10A-GFP in top andbottom regions of the fiber bundle. The results showed there weresignificantly more MCF-7-RFP cells in the top region of the fiber bundlethan MCF-10A-GFP cells. There was no significant difference in cellcount between cell types in the bottom region of the fiber.

To quantify the separation the fibers were sectioned in half with ablade creating a top and bottom region. Each region was transferred to anew empty well, washed with phosphate-buffered saline, unwound withforceps, and soaked in Trypsin-EDTA solution to remove the cells. Thecell count in each region of the fiber was determined using flowcytometry (Guava Technologies) with InCyte software (GuavaTechnologies). Both the complexity and size of the cells and fluorescentprotein expression were used to quantify the number of cancerous andbenign cells in each region. FIG. 11 illustrates the percentage of eachcell type in top and bottom regions demonstrating a significantdifference (p<0.05) between percentage of MCF-7-RFP and MCF-10A-GFP.These results make evident that the wicking fiber bundle can separateand isolate cancerous cells from a mixture with high purity, as thegraph indicates 82% of cells isolated from top region are cancerous.

Example 3

To distinguish progenitor cells from a further differentiated cell linethe following cell lines were used: D1 mouse mesenchymal stem cells(ATCC) and RAW mouse macrophage cells (ATCC). D1 and Raw cells werelabeled with fluorescent probes, CellTracker Red CMTPX (Invitrogen) andCellTracker Green CMFDA (Invitrogen), respectively. A device and fiberbundles as described above in Example 1 were used. Both cell types wereplaced in each of the wells of a low attachment 12-well plate at adensity of one million per mL. The fibers were vertically inserted intothe custom-made lids designed to keep the fiber vertical and only 3 mmof the tip submersed. The displacement of the D1 and raw cells wasdetermined at time points of 1 and 24 hours using fluorescent microscopyand imaging software. ImageJ was used to evaluate the cellulardisplacement and density of each cell type.

Results are shown in FIG. 12. As can be seen, D1 cells exhibited greatervertical displacement and cell densities along the fiber than the RAWcells.

Example 4

To evaluate the effect of an absorptive cap on the fluid transport ofwicking fibers a vertical test with dye-solution was performed forsamples containing only a bundle of three braided wicking fibers andsamples containing a bundle of three braided wicking fibers with aglobule of alginate on one end. The fluid transport of dye-solution insingle wicking fibers and single wicking fibers with alginate is alsoevaluated. 1-mL of dye-solution was pipetted into each well of a 12-wellplate. Three samples 4-cm in length containing only braided wickingfibers and three samples 4-cm in length with alginate were verticallyplaced into each of the wells. The end of the wicking fiber notcontaining the alginate was placed into the dye-solution of the well, asshown in FIG. 13. Three single fibers 4-cm in length and three singlefibers containing alginate globules were also vertically placed in thedye-solution.

Each well of the 12-well plate initially contained 1-mL of dye-solution.After 24 hours of the vertical test the amount of volume left in thewell was measured using a pipettor. The change in volume in the wellbefore and after the vertical test was recorded as the volume absorbedas shown in FIG. 14. This chart quantitatively shows bundled fibersenhance the amount of liquid transported and absorbed compared to singlefibers. The results also indicate that alginate increases the amount ofliquid absorbed by the braided and single fibers. Students T-test showsa significant difference (*p<0.05) between each fiber set-up (i.e.braided or single) with and without alginate.

Example 5

A vertical test was conducted with a cell solution to evaluate themovement of cells through a device. The cell-solution contained two celltypes, normal and cancer cells, of equal cell density of 500,000cells/mL. The normal cell line and cancer cell line were labeled withgreen and red fluorescents, respectively, to analyze the verticalmovement of different cell types. The test was performed for twelvesamples containing a bundle of three braided wicking fibers and aglobule of alginate on one end. 1 mL of cell-solution containing bothcell types was pipetted into each well of a low-attachment 12-wellplate. Four samples were imaged after the respected time points of 3hours, 24 hours, and 48 hours taken from the experimental set-up.

After 24 hours the alginate was removed from each sample and dissolvedallowing for the isolation of normal and cancer cells from the sample. Ahemocytometer was used to count the amount of cancer and normal cells inthe alginate components for each of the samples. FIG. 15 illustrates theresults and shows the cancer cells collecting in the alginate moresignificantly than the normal cells (p<0.01).

The results indicate the alginate wicking fiber construct can evaluatecell types and function as a diagnostic tool based on the verticalmovement in the fibers and collection of cells in the alginate.

The amount of fibers and alginate used can increase the volume of fluidand cells transported. In another run, a bundle of 20 fibers and avolume of alginate five times the amount used previously were utilized.As a result, more fluid and cells traveled through the fibers andalginate in a shorter amount of time. The alginate collectedapproximately 10 times the amount of cancer cells as recorded in theprevious run.

Example 6

The transport properties of constructs including channeled polylactidefibers with an absorbent alginate cap, channeled polylactide fiberswithout the cap, and round polylactide fiber constructs were evaluated.The vertical wicking rates of these constructs were determined byanalyzing the change in height of the liquid front over time. FIG. 16Aillustrates the wicking rate of the capped constructs compared to theround and uncapped constructs. As can be seen, the capped channeledfiber constructs had enhanced fluid transport properties. The rate ofthe fluid front moving vertically was significantly greater in thecapped construct than in round fiber or uncapped fiber constructs.

Bone progenitor cell penetration and retention was assessed in thecapped fiber constructs and uncapped fiber constructs by assessing thevertical movement and cell densities of green-labeled D1 mousemesenchymal progenitor cells (CellTracker green probe; Invitrogen) byfluorescent microscopy and quantifying cell movement along theconstructs using Guava EasyCyte™ flow cytometry (Guava Technologies).Results are illustrated in FIG. 16B. As can be seen, there wassignificantly greater cellular recruitment into the top region of thecapped constructs than into round or uncapped constructs. (Asterisksindicate significant differences (p<0.05).)

Capped channeled fiber constructs were also incorporated into chronOS®strip scaffolds and seeded using custom-made vacuum sealed perfusionpacks. Samples were incubated at room temperature for 1 hour. Cellinfiltration, distribution, viability, and proliferation into thescaffolds were assessed by (1) imaging fluorescent probes DAPI andphalloidin (Invitrogen) in the center and peripheral regions of thescaffold (2) Live/Dead cytotoxicity assay (Invitrogen) and Viacountassay using Guava flow cytometer, (3) PicoGreen Assay (Invitrogen) andbiochemistry analyzer (YSI 2700). Results are shown in FIG. 16C. As canbe seen, the scaffold-modified construct increased both overall cellcount and number of viable cells as compared to the scaffold alone.(Asterisks indicate significant differences (p<0.05).) The results alsoshowed that the central interior region of the modified scaffold hadenhanced cellular infiltration and distribution as well as significantlymore viable cells and greater proliferation.

Example 7

The transport properties of a channeled fiber construct modified byinclusion of an absorbent alginate cap as described above and anunmodified channeled fiber construct were evaluated by comparing therate of wicking and amount of fluid absorbed over time. The verticalwicking rate was determined by analyzing the change in height of theliquid front over time after placing the samples vertically in a wellcontaining dye-solution. The volume of absorbed fluid was determined bymeasuring the amount of fluid remaining in the well. FIG. 17Ademonstrates the volume of fluid absorbed in the modified and unmodifiedconstructs. (Asterisks indicate significant differences (p<0.05).) Ascan be seen, the modified construct showed enhanced fluid transportproperties. The rate of the fluid front moving vertically wassignificantly greater in the alginate-capped samples. As shown, theamount of fluid absorbed after 12 hours and 24 hours was greater in thealginate-capped constructs as compared to the unmodified constructs.

The alginate-capped construct was used to separate and isolate cancerousmammary epithelial cells, MCF-7 (ATCC), from a mixture of benign mammaryepithelial cells MCF-10A (ATCC) and MCF-7 cells. To track the separationof the cell lines, benign and cancerous cells were stably transfectedwith Green Fluorescent Protein (GFP) or Red Fluorescent Protein (RFP),respectively. The separation along the construct was evaluated at 3hours, 24 hours, and 48 hours with fluorescent microscopy. After 24hours the cells were removed from the constructs and the percentage ofbenign and cancerous cells were determined using Guava EasyCyte™ flowcytometry (Guava Technologies) by evaluating the percentage of red andgreen fluorescent count. Positive and negative controls of known celldensities were used to calibrate the machine before measuring thetreatment groups.

Fluorescent images of MCF7-RFP and MCF10A-GFP demonstrated separation ofcancerous cells along the construct. FIG. 17B depicts a high percentageof cancerous MCF7-RFP cells isolated from the constructs after 24 hours.(Asterisks indicate significant differences (p<0.05).)

Results indicate the alginate-capped construct showed enhanced transportproperties that can be used for cell separation of normal andpathological cells. The results suggest the device can separate andisolate pathological cells from a mixture of cells in solution with highpurity and efficiency. This device provides a rapid and label-freeapproach to isolate various cell types for pathological tissue testsystem applications.

It will be appreciated that the foregoing examples, given for purposesof illustration, are not to be construed as limiting the scope of thisdisclosure. Although only a few exemplary embodiments of the disclosedsubject matter have been described in detail above, those skilled in theart will readily appreciate that many modifications are possible in theexemplary embodiments without materially departing from the novelteachings and advantages of this disclosure. Accordingly, all suchmodifications are intended to be included within the scope of thisdisclosure. Further, it is recognized that many embodiments may beconceived that do not achieve all of the advantages of some embodiments,yet the absence of a particular advantage shall not be construed tonecessarily mean that such an embodiment is outside the scope of thepresent disclosure.

What is claimed is:
 1. A diagnostic device comprising: a well that isconfigured for holding a fluid sample; a capillary channeled fiberhaving a longitudinal axis along a length of the fiber an outer surface,and a non-circular cross section that is perpendicular to thelongitudinal axis of the fiber, the capillary channeled fiber includinga plurality of co-linear channels extending along the entire length ofthe fiber, each channel being open at the outer surface of the fiber,each channel being defined by two opposed walls, each wall thatextending in a first direction that is along the length and parallel tothe longitudinal axis of the fiber and each wall also extending in asecond direction that is perpendicular to the longitudinal axis of thefiber with each wall forming a side wall of one channel and each wallforming a part of the exterior surface of the fiber; and a brace locatedin or above the well, the brace comprising a structure that secures thecapillary channeled fiber in a vertical orientation with a first end ofthe fiber within the well.
 2. The diagnostic device of claim 1, whereinthe brace is a lid configured to cover a top of the well, the lidincluding an aperture that passes from a first side of the lid to asecond side of the lid, the aperture being configured to secure thefiber.
 3. The diagnostic device of claim 1, wherein the capillarychanneled fiber is a member of a bundle of fibers that are held adjacentto one another and generally aligned with one another in the bundle. 4.The diagnostic device of claim 3, wherein the fibers of the bundle areheld adjacent to one another and generally aligned with one another suchthat at least one closed channel is formed between two adjacentcapillary channeled fibers of the bundle.
 5. The diagnostic device ofclaim 1, wherein the device further includes a cap that covers and isattached to a second end of the capillary channeled fiber with thesecond end of the capillary channeled fiber enclosed within the cap, thecap including a sorptive material that surrounds the second end of thecapillary channeled fiber.
 6. A method for separating biologicalmaterials by use of the device of claim 1, the method comprising:locating a fluid sample that includes a first biological material and asecond biological material within the well, the first biologicalmaterial wicking through the channels of the capillary channeled fiberto a first location of the device during a period of time and the secondbiological material wicking through the channels of the capillarychanneled fiber to a second location of the device during the period oftime; and recovering at least one of the first biological material orthe second biological material sell from the device.
 7. The method ofclaim 6, wherein the first biological material is a cancer cell and thesecond biological material is a healthy cell, the two cells being of thesame cell type.
 8. The method of claim 7, wherein the two cells arebreast epithelial cells.
 9. The method of claim 6, wherein the firstbiological material is a progenitor cell and the second biologicalmaterial is a differentiated cell.
 10. The method of claim 9, whereinthe first biological material is a mesenchymal stem cell.
 11. The methodof claim 9, wherein the second biological material is a macrophage cell.12. The diagnostic device of claim 5, wherein the sorptive material isan adsorptive material.
 13. The diagnostic device of claim 5, whereinthe sorptive material in an absorptive material.
 14. The diagnosticdevice of claim 5, the cap comprising an alginate.
 15. The diagnosticdevice of claim 5, the cap comprising a coating that includes ahydrophobic or water impermeable material.
 16. The diagnostic device ofclaim 1, wherein the width of each of the co-linear channels is lessthan about 0.5 millimeters.
 17. The diagnostic device of claim 1,wherein the capillary channeled fiber is a polymeric fiber.
 18. Thediagnostic device of claim 17, the polymeric fiber comprising abiocompatible polymer.
 19. The diagnostic device of claim 1, thecapillary channeled fiber comprising a biologically active agent withinor on the surface of the fiber.